IDENTIFICATION OF A RESIDUE IN THE TRANSLOCATION PATHWAY OF A MEMBRANE CARRIER

Preliminary work using directed mutagenesis proved that cysteine is no t required for operation of UhpT, the anion exchange protein responsib le for glucose 6-phosphate transport by E. coll. We then made a detail ed study of C143 and C265, because these cysteines impart sensitivity to p-chloromercuribenzosulfonate (PCMBS), a sulfhydral agent resemblin g glucose 6-phosphate in size, shape, and charge. We showed that C143 was exposed to the cytoplasm, as expected from hydropathy analysis, bu t we found no sidedness for C265. Rather, C265 was accessible to PCMBS from both membrane surfaces. And since the attack at C265 was blocked by glucose 6-phosphate, position 265 must lie directly on the pathway taken by the substrate as it moves through this membrane carrier.